Hi All,
The kit is Pierce's CarboLink; I didn't really know if it's conventional to
mention those details here or not.
The gel itself doesn't bind the enzyme-linked test protein to a significant
extent. Until that is the gel has been exposed to antibody to the test
protein, whether the antibody's been pre-treated with Pierce's periodate
reagent or not. Strangely (in my opinion), pre-heating the antibody to 37C
works about as well as pre-treating it with the periodate.
I guess the antibody could be denatured by heating, made stickier to the gel
and yet still specific to its ligand, but that seems a stretch.
It's an excellent point, but an irrelevant antibody doesn't confer affinity
to the test protein (which is a mouse anti Rabbit~HRP conjugate).
I have to admit that I don't know how much protein antibody the gel is
taking up, because I haven't been measuring the content of what passes
through when that incubation is over. I have done a BCA on the gels from
the columns. The CarboLink gel itself is apparently reactive to the BCA
reagent, so it's hard to say how much protein is there.
I've tried Pierce tech support and been assured that the gel is passivated
enough, but I may try them again.
The other thing that occurs to me to try is tests with something like an
anti BSA so I can use BSA for the ligand, and see if any of these
preparations is allowing me to bind much of the test protein.
I've been trying pretty hard to make this work, and I appreciate your input;
have a good one :)
DanG
-----Original Message-----
From: proteins-bounces from oat.bio.indiana.edu
[mailto:proteins-bounces from oat.bio.indiana.edu] On Behalf Of
proteins-request from oat.bio.indiana.edu
Sent: Saturday, March 31, 2007 12:01 PM
To: proteins from magpie.bio.indiana.edu
Subject: Proteins Digest, Vol 22, Issue 15
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Today's Topics:
1. Re: affinity columns (Allison)
2. RE: RE: Re: affinity columns (Allison) (DK) (Dan Guire)
----------------------------------------------------------------------
Message: 1
Date: Fri, 30 Mar 2007 16:40:06 -0500
From: Allison <allison from nospam.com>
Subject: [Protein-analysis] Re: affinity columns
To: proteins from net.bio.net
Message-ID: <nrmdnXrKZOY26JDbnZ2dnUVZ_sWdnZ2d from mcgill.ca>
Content-Type: text/plain; charset=us-ascii; format=flowed
Dan Guire wrote:
> Hi Allison,
>> I'm trying to prepare an immobilized-antibody affinity column. I've tried
> using a commercial kit that includes a gel which is intended to bind
> antibodies that have been pretreated with a reagent that's included in the
> kit. My problem is that in the course of troubleshooting the procedure, I
> found that just exposing the gel to the antibody solution without
> pretreating the antibody with their reagent is enough to confer affinity
to
> the gel. It binds my antigen about as well as if I had used their
reagent,
> so the antibody must be passively adsorbed there to a significant extent.
>> Columns prepared in this way can stand up to acid elutions and salt washes
> about as well too. So my question is: Is it possible that it's not
> preferable to covalently immobilize the affinity agent? I doubt that,
which
> leads me to think that my method is misleading me.
>> In testing the kit I've bought I've been using an enzyme-labeled protein
as
> ligand. Since a small amount of enzyme can give back a large signal, the
> total amount of protein is small. Maybe I haven't been loading enough
> ligand onto the columns to reflect the true amounts of immobilized
antibody
> on the differently-prepared columns.
>> Hey I might try doing a BCA on the gels themselves to see if there's more
> protein on one than the other, or using a larger loading of an unlabeled
> version of my ligand. Any other thoughts will be much appreciated,
>> DanG
>
I haven't made my own affinity columns but some questions do come to mind:
Does your gel alone bind the test protein or does an Ab with a different
specificity also give the same effect? Because I agree with you that
enzyme-labelled proteins can sometimes be much too sensitive. Perhaps
the gel has some inherent "stickiness" and will bind a bit of Ab and a
bit of ligand without any treatment at all.
Does the kit come with any info as to how much antibody can be coupled
per gm of gel? If so, how does this compare with how much you can bind
without pretreatment.
What does the company that makes the kit say when you contact them?
Presumably they have lots of experience with troubleshooting and it is
in their best interests to help solve your problem for you.
Do you want to let us know the name of kit/company?
Allison
------------------------------
Message: 2
Date: Fri, 30 Mar 2007 17:35:06 -0500
From: "Dan Guire" <dguire from isurtec.com>
Subject: [Protein-analysis] RE: RE: Re: affinity columns (Allison)
(DK)
To: <proteins from oat.bio.indiana.edu>, <proteins from magpie.bio.indiana.edu>
Message-ID: <000301c7731b$aaf71820$4f6fa8c0 from IsurTec.local>
Content-Type: text/plain; charset="us-ascii"
Hi DK,
Yes it's a hydrazide-functional beaded agarose gel support, and the antibody
or other glycoprotein is oxidized with periodate to form reactive aldehydes.
I've conferred enough antibody activity to the gel without using the
periodate reagent to make me doubt that the principle of the kit's
preparation is sound. Direct ELISAs are based on the stickiness of protein,
maybe the antibody just sticks to the agarose whether the reagent is used or
not.
DanG
-----Original Message-----
From: proteins-bounces from oat.bio.indiana.edu
[mailto:proteins-bounces from oat.bio.indiana.edu] On Behalf Of
proteins-request from oat.bio.indiana.edu
Sent: Friday, March 30, 2007 12:02 PM
To: proteins from magpie.bio.indiana.edu
Subject: Proteins Digest, Vol 22, Issue 14
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Today's Topics:
1. Re: Enthalpy of protein (Protenger)
2. Re: Enthalpy of protein (Frank K?ster)
3. RE: Re: affinity columns (Allison) (Dan Guire)
4. RE: Re: affinity columns (Allison) (DK)
----------------------------------------------------------------------
Message: 1
Date: 29 Mar 2007 08:53:44 -0700
From: "Protenger" <yellowish from gmail.com>
Subject: [Protein-analysis] Re: Enthalpy of protein
To: proteins from net.bio.net
Message-ID: <1175183624.882753.238960 from p15g2000hsd.googlegroups.com>
Content-Type: text/plain; charset="iso-8859-1"
On Mar 29, 1:20 pm, Frank K|ster <f... from kuesterei.ch> wrote:
> "Protenger" <yellow... from gmail.com> wrote:
> > I ask my question in another format.
> > Suppose we have a native protein with its own intristic (absolute)
> > enthalpy, then by protein engineering we introduce a pair
> > of opposide charged residues on its surface so its stability
> > increases.
> > Does absolut (and not delta H) enthalpy of mutant decrease?
>> What is absolute enthalpy, and what is it good for to know it?
>> Regards, Frank
> --
> But this:> For fucks sake...
>> is just offensive. It should have an apostrophe(!)
> [MJ Ray, nowhere]
One of my fellows said "by introducing stabilizing interaction in
proteins (in its core or surface) its enthalpy will increase"
This sentence have confused me very much, because as I have learned
before reactions (for example protein folding) tend to
reduce their enthalpy by releasing heat so stabilized protein (native)
relative to intermediate one (for example molten globule state) must
have a lower enthalpy.
So what about engineered proteins that stabilized for example by
introduced salt bridges?
What is the deference between intrinsic (absolute) enthalpy between
them?
Or clearly, which of them has higher enthalpy?
Regards, Rahim
------------------------------
Message: 2
Date: Thu, 29 Mar 2007 18:59:57 +0200
From: Frank K?ster <frank from kuesterei.ch>
Subject: [Protein-analysis] Re: Enthalpy of protein
To: proteins from net.bio.net
Message-ID: <dt7td4-r58.ln1 from riesling.kuesterei.ch>
Content-Type: text/plain; charset=us-ascii
"Protenger" <yellowish from gmail.com> wrote:
> One of my fellows said "by introducing stabilizing interaction in
> proteins (in its core or surface) its enthalpy will increase"
That doesn't sound correct. First of all, as has been pointed out, the
absolute enthalpy is both hard to get and quite boring. The interesting
question, therefore: Which process is considered? If it is complete
unfolding, then the quantity of interest is the enthalpy of
folding/unfolding.
> This sentence have confused me very much, because as I have learned
> before reactions (for example protein folding) tend to
> reduce their enthalpy by releasing heat so stabilized protein (native)
> relative to intermediate one (for example molten globule state) must
> have a lower enthalpy.
Err, are we talking about enthalpy or Gibbs (Free) enthalpy?
And what is it that confuses you? Are you sure you were both talking
about the same process? Naturally, if the (Gibbs) enthalpy of unfolding
decreases upon a mutation, the (Gibbs) enthalpy of folding will
increase.
> So what about engineered proteins that stabilized for example by
> introduced salt bridges?
> What is the deference between intrinsic (absolute) enthalpy between
> them?
> Or clearly, which of them has higher enthalpy?
Why are you interested in absolute enthalpies, when all that is
experimentally accessible is the change in enthalpy (or Gibbs enthalpy)
associated with a process?
Regards, Frank
--
But this:
> For fucks sake...
is just offensive. It should have an apostrophe(!)
[MJ Ray, nowhere]
------------------------------
Message: 3
Date: Thu, 29 Mar 2007 13:34:31 -0500
From: "Dan Guire" <dguire from isurtec.com>
Subject: [Protein-analysis] RE: Re: affinity columns (Allison)
To: <proteins from oat.bio.indiana.edu>
Message-ID: <000101c77230$e480ee40$4f6fa8c0 from IsurTec.local>
Content-Type: text/plain; charset="us-ascii"
Hi Allison,
I'm trying to prepare an immobilized-antibody affinity column. I've tried
using a commercial kit that includes a gel which is intended to bind
antibodies that have been pretreated with a reagent that's included in the
kit. My problem is that in the course of troubleshooting the procedure, I
found that just exposing the gel to the antibody solution without
pretreating the antibody with their reagent is enough to confer affinity to
the gel. It binds my antigen about as well as if I had used their reagent,
so the antibody must be passively adsorbed there to a significant extent.
Columns prepared in this way can stand up to acid elutions and salt washes
about as well too. So my question is: Is it possible that it's not
preferable to covalently immobilize the affinity agent? I doubt that, which
leads me to think that my method is misleading me.
In testing the kit I've bought I've been using an enzyme-labeled protein as
ligand. Since a small amount of enzyme can give back a large signal, the
total amount of protein is small. Maybe I haven't been loading enough
ligand onto the columns to reflect the true amounts of immobilized antibody
on the differently-prepared columns.
Hey I might try doing a BCA on the gels themselves to see if there's more
protein on one than the other, or using a larger loading of an unlabeled
version of my ligand. Any other thoughts will be much appreciated,
DanG
-----Original Message-----
From: proteins-bounces from oat.bio.indiana.edu
[mailto:proteins-bounces from oat.bio.indiana.edu] On Behalf Of
proteins-request from oat.bio.indiana.edu
Sent: Thursday, March 29, 2007 12:01 PM
To: proteins from magpie.bio.indiana.edu
Subject: Proteins Digest, Vol 22, Issue 13
Send Proteins mailing list submissions to
proteins from net.bio.net
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When replying, please edit your Subject line so it is more specific
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Today's Topics:
1. Re: affinity columns (Allison)
2. Re: Enthalpy of protein (Raoul Fleckman)
3. Re: Enthalpy of protein (Frank K?ster)
----------------------------------------------------------------------
Message: 1
Date: Wed, 28 Mar 2007 15:27:46 -0500
From: Allison <allison from nospam.com>
Subject: [Protein-analysis] Re: affinity columns
To: proteins from net.bio.net
Message-ID: <qeadnT45_8AqXJfbnZ2dnUVZ_sjinZ2d from mcgill.ca>
Content-Type: text/plain; charset=us-ascii; format=flowed
Dan Guire wrote:
> Hi Barbara,
>> The main point has already been made: Your anti rabbit alphatase
conjugate
> can only detect the rabbit antibody down on the Maxisorp plate. It
doesn't
> matter whether there is antigen for the capture antibody or not. No doubt
a
> negative control, leaving out only the mouse lysate, would show you that.
>> The series Allison outlined looks fine; the enzyme-conjugated anti mouse
> shouldn't bind unless there is mouse antibody bound to antigen there. My
> point is that if you already have biotinylated anti ENAC of one species,
you
> can capture any antigen with the non-biotinylated anti ENAC of the other
> species, then detect with the biotin and a streptavidin-enzyme conjugate.
> And a negative control is a must :-)
>> DanG
>> P.S. Do you have any affinity column experience by any chance? I could
use
> some help in that arena...
>>
I have done a bit....what is your question? If I can't help I am sure
someone else can.
Allison
------------------------------
------------------------------
Message: 4
Date: Fri, 30 Mar 2007 03:37:34 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: [Protein-analysis] RE: Re: affinity columns (Allison)
To: proteins from net.bio.net
Message-ID: <3C%Oh.2214$qe1.1608 from newsfe04.lga>
In article <mailman.563.1175200089.5139.proteins from net.bio.net>, "Dan Guire"
<dguire from isurtec.com> wrote:
>Hi Allison,
>>I'm trying to prepare an immobilized-antibody affinity column. I've tried
>using a commercial kit that includes a gel which is intended to bind
>antibodies that have been pretreated with a reagent that's included in the
>kit. My problem is that in the course of troubleshooting the procedure, I
>found that just exposing the gel to the antibody solution without
>pretreating the antibody with their reagent is enough to confer affinity to
>the gel. It binds my antigen about as well as if I had used their reagent,
>so the antibody must be passively adsorbed there to a significant extent.
>>Columns prepared in this way can stand up to acid elutions and salt washes
>about as well too. So my question is: Is it possible that it's not
>preferable to covalently immobilize the affinity agent? I doubt that,
which
>leads me to think that my method is misleading me.
Knowing what is in the kit anf how it works can do wonders to
understanding what's going on and troubleshooting. Do they tell you the
principle and the chemistry involved?
DK
------------------------------
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